RESEARCH ARTICLE
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*For correspondence: lora.
hooper@utsouthwestern.edu
†
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competing interests exist.
Funding: See page 15
Received: 30 April 2014
Accepted: 28 July 2014
Published: 29 July 2014
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Derebe et al. eLife 2014;3:e03206. DOI: 10.7554/eLife.03206 1 of 18
Serum amyloid A is a retinol binding
protein that transports retinol during
bacterial infection
Mehabaw G Derebe1†, Clare M Zlatkov1†, Sureka Gattu1, Kelly A Ruhn1,
Shipra Vaishnava1, Gretchen E Diehl2, John B MacMillan3, Noelle S Williams3,
Lora V Hooper1,4*
1
Department of Immunology, University of Texas Southwestern Medical Center,
Dallas, United States; 2Molecular Pathogenesis Program, The Kimmel Center for
Biology and Medicine of the Skirball Institute, New York University School of
Medicine, New York, United States; 3Department of Biochemistry, University of
Texas Southwestern Medical Center, Dallas, United States; 4Howard Hughes Medical
Institute, University of Texas Southwestern Medical Center, Dallas, United States
Abstract Retinol plays a vital role in the immune response to infection, yet proteins that mediate
retinol transport during infection have not been identified. Serum amyloid A (SAA) proteins are
strongly induced in the liver by systemic infection and in the intestine by bacterial colonization,
but their exact functions remain unclear. Here we show that mouse and human SAAs are retinol
binding proteins. Mouse and human SAAs bound retinol with nanomolar affinity, were associated
with retinol in vivo, and limited the bacterial burden in tissues after acute infection. We determined
the crystal structure of mouse SAA3 at a resolution of 2 Å, finding that it forms a tetramer with a
hydrophobic binding pocket that can accommodate retinol. Our results thus identify SAAs as a
family of microbe-inducible retinol binding proteins, reveal a unique protein architecture involved in
retinol binding, and suggest how retinol is circulated during infection.
DOI: 10.7554/eLife.03206.001
Introduction
Retinol plays a vital role in the physiological response to microbial challenge. Retinol is derived from
dietary vitamin A and can be converted enzymatically to retinoic acid, which complexes with nuclear
receptors to regulate gene transcription programs in cells (Germain et al., 2006). In this way, retinol
promotes the maturation of innate immune cells (Lawson and Berliner, 1999; Stephensen, 2001;
Spencer et al., 2014), governs the differentiation of adaptive immune cells (Mucida et al., 2007;
Hall et al., 2011), and facilitates the regeneration of epithelial barriers damaged by infection (Osanai
et al., 2007). A hallmark of vitamin A deficiency in humans is a markedly increased susceptibility to
infection (Sommer 2008, Underwood, 2004), underscoring the broad impact of retinol on immunity.
As a small lipid-soluble compound, retinol cannot freely circulate but is instead transported among
cells and tissues by specialized retinol binding proteins. Serum retinol binding protein (RBP) facilitates
transport of retinol among the intestine, which is the site of retinol acquisition, the liver, which is the
major site of retinoid storage, and other tissues that require retinol for their physiological functions
(Blaner, 1989). Despite the increased requirement for retinol, serum RBP is markedly reduced fol-
lowing microbial challenge (Rosales et al., 1996), leaving open the question of how retinol is trans-
ported among tissues during infection.
Serum amyloid A (SAA) proteins are a family of proteins that are expressed in the intestinal epithe-
lium (Eckhardt et al., 2010; Reigstad and Bäckhed, 2010) and liver (Uhlar and Whitehead, 1999)
 Research article Biophysics and structural biology | Immunology

eLife digest Vitamins are nutrients that organisms require in order to survive and grow. If an
organism is unable to synthesize a vitamin in sufficient quantities, it is essential that it obtain the
vitamin through its diet instead.
Vitamin A is found in foods such as eggs, animal liver and carrots, and a diet that is lacking in this
vitamin can cause blindness and an increased risk of microbial infections. Vitamin A is not a single
compound, but rather a collection of compounds with similar molecular structures. One of these is
retinol, which plays a vital role in the body's response to microbial infection. Retinol must bind to
specific proteins to be able to move through the bloodstream and be transported around the body.
Serum retinol binding protein transports ingested retinol from the intestine to the liver and other
tissues. However, during microbial infection—when retinol transport is particularly important—the
amount of this protein dramatically decreases; as such it is unclear how retinol is transported when
the body is under attack from pathogens.
It had been suggested that Serum Amyloid A (SAA) proteins, a family of proteins made by some
liver and intestinal cells, could be involved in the response to infection, because these proteins'
levels increase during infection. However, their exact functions were unknown. Derebe, Zlatkov
et al. found that mice fed a diet poor in vitamin A produced fewer SAA proteins in their liver and
intestinal cells. However, treating the cells with retinol or the molecule it is broken down into—
called retinoic acid—caused more SAAs to be made. Derebe, Zlatkov et al. also discovered that
SAAs are associated with retinol in blood samples taken from mice infected with salmonella; and
that both mouse and human SAAs bind tightly to retinol. Combined, this evidence suggests that
SAAs are the retinol binding proteins that transport retinol during infections.
Derebe, Zlatkov et al. went on to solve the crystal structure of a mouse SAA protein, and showed
that four SAA molecules bind together to form a ‘pocket’ that can hold a retinol molecule. Future
work will focus on understanding exactly how the transport of retinol by SAAs affects the
development of immunity to infections.
DOI: 10.7554/eLife.03206.002

and circulate in the serum (Whitehead et al., 1992). SAA family members are encoded in the genomes
of virtually all vertebrates and are highly conserved among species, suggesting essential biological
functions (Uhlar et al., 1994). Expression of SAAs is strongly induced by microbial exposure. SAAs are
induced in intestinal epithelial cells by the microbiota (Ivanov et al., 2009; Reigstad et al., 2009;
Eckhardt et al., 2010; Reigstad and Bäckhed, 2010), and have been implicated in promoting Th17
cell development in response to specific microbiota components (Ivanov et al., 2009). Similarly, liver
and serum SAAs are markedly elevated following systemic bacterial or viral infection (Meek and
Benditt, 1986; Chiba et al., 2009).
Although it has been proposed that SAAs generally contribute to inflammation and immunity
(Eckhardt et al., 2010), the exact functions of SAAs remain poorly defined. Interestingly, SAAs have
characteristics that suggest they could bind hydrophobic ligands. First, all SAAs are predicted to form
amphipathic helices with a hydrophobic face that could interact with non-polar molecules (Stevens,
2004). Second, SAAs circulate in the serum associated with high-density lipoprotein (HDL), which
transports lipid-bound lipoproteins amongst tissues (Whitehead et al., 1992). However, the identity
of potential SAA ligand(s) remains unclear.
Here, we show that mouse and human SAAs are retinol binding proteins. We demonstrate that SAA
expression in mice requires dietary vitamin A, that mouse and human SAAs bind tightly to retinol, and
that SAA recovered from serum following bacterial infection is associated with retinol. We find that
Saa1/2−/− mice, which harbor deletions of both the Saa1 and Saa2 genes, have higher bacterial bur-
dens in spleen and liver following an acute bacterial infection, supporting an essential role for SAAs in
the response to microbial challenge. Finally, we provide structural insight into the binding interaction
by solving the mouse SAA3 crystal structure, which reveals a tetrameric assembly with a hydrophobic
binding pocket that can accommodate retinol. These studies thus identify SAAs as a family of retinol
binding proteins and reveal a new protein architecture supporting retinol binding. Our findings sug-
gest that SAAs mediate retinol transport during microbial challenge and thus constitute a key compo-
nent of the physiological response to infection.

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 Research article Biophysics and structural biology | Immunology

Results
SAA expression requires dietary vitamin A
Initially we uncovered a relationship between SAA expression and dietary vitamin A status in mice.
Microarray analysis disclosed that mice fed a vitamin A-deficient diet exhibited lower abundances of
serum amyloid A (Saa) 1 and 2 transcripts in the intestine as compared to mice fed a vitamin A-replete
diet (Figure 1—figure supplement 1; Table 1). Real-time quantitative PCR and immunofluorescence
analysis verified that expression of small intestinal SAA1, 2, and 3 was reduced in mice fed a vitamin
A-deficient diet (Figure 1A,B; Table 1). Liver expression of SAA1 and 2 was also reduced in mice fed
a vitamin A-deficient diet, although the reduction in expression was less pronounced than in the intes-
tine (Figure 1C,D). This is likely because dietary vitamin A deficiency does not completely deplete
stored retinoids in the liver (Liu and Gudas, 2005). We also observed elevated expression of intestinal
Saa1 and Saa2 following addition of retinol directly to the epithelial surface of small intestinal explants,
and of liver Saa1 and Saa2 after intraperitoneal supplementation with retinoic acid (Figure 1—figure
supplement 2). These findings support the idea that retinoids directly impact Saa expression. Addition
of retinol or retinoic acid to cultured HepG2 cells (a human liver cell line) enhanced expression of SAA1
and 2 in the presence of IL-1β and IL-6 (Figure 1E,F), suggesting that the impact of dietary vitamin A
on SAA expression is due to cell-intrinsic effects of retinoids. Collectively, these results show that full
expression of SAAs in the intestine and liver requires dietary vitamin A.

Human and mouse SAAs bind

Table 1. Primers used in Q-PCR analysis
retinol
Primer Transcriptional control by retinoids is frequently
name Primer sequence observed in proteins that function in retinoid trans-
mouse 5ʹ-CATTTGTTCACGAGGCTTTCC port and metabolism (Noy, 2000). Because SAAs
SAA1 F have predicted hydrophobic binding surfaces
mouse 5ʹ-GTTTTTCCAGTTAGCTTCCTTCATGT (Stevens, 2004) and are induced by retinol, we
SAA1 R hypothesized that they might be retinol binding
mouse 5ʹ-TGTGTATCCCACAAGGTTTCAGA proteins. We therefore tested for retinol binding
SAA2 F
activity of recombinant human SAA1 (hSAA1),
mouse 5ʹ-TTATTACCCTCTCCTCCTCAAGCA mouse SAA1 (mSAA1), and mouse SAA3 (mSAA3)
SAA2 R
using fluorometric binding assays that exploit
mouse 5ʹ-CGCAGCACGAGCAGGAT the unique spectral properties of retinol. (Note
SAA3 F
that we were unable to express recombinant
mouse 5ʹ-CCAGGATCAAGATGCAAAGAATG
SAA3 R
mSAA2). Retinol exhibits intrinsic fluorescence
that is enhanced upon binding to proteins through
human 5ʹ-GGCATACAGCCATACCATTC
SAA1 F energy transfer from tryptophan residues, and
this fluorescence change can be used to quantify
human 5ʹ-CCTTTTGGCAGCATCATAGT
SAA1 R binding (Cogan et al., 1976) (Figure 2A). We did
human 5ʹ-GCTTCCTCTTCACTCTGCTCT
fluorometric titrations to determine apparent dis-
SAA2 F sociation constants (Kds) for the all-trans isomer
human 5ʹ-TGCCATATCTCAGCTTCTCTG of retinol, and extracted Kds of 259, 169, and
SAA2 R 145 nM for retinol binding to hSAA1, mSAA1,
mouse 5ʹ-CATTCGAACGTCTGCCCTATC and mSAA3, respectively (Figure 2B,E). These
18S F values are similar to binding affinities calculated
mouse 5ʹ-CCTGCTGCCTTCCTTGGA for human serum retinol binding protein (hRBP)
18S R (Cogan et al., 1976) (Figure 2—figure supple-
mouse 5ʹ-TGGCAAAGTGGAGATTGTTGCC ment 1). Thus, SAAs bind retinol tightly, with
Gapdh F affinities similar to that of a known retinol binding
mouse 5ʹ-AAGATGGTGATGGGCTTCCCG protein.
Gapdh R
Retinoic acid lacks intrinsic fluorescence but
human 5ʹ-CCTGGTCACCAGGGCTGCTTTTAAC can quench inherent protein fluorescence due to
Gapdh F
energy transfer from tryptophan residues (Cogan
human 5ʹ-GTCGTTGAGGGCAATGCCAGCC et al., 1976). We therefore measured retinoic acid
Gapdh R
binding using a modified fluorescence assay that
DOI: 10.7554/eLife.03206.003 monitored quenching of protein fluorescence

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Figure 1. SAA expression requires dietary vitamin A. (A) Mice were maintained on a normal (Vit. A+) diet or a
vitamin A-deficient (Vit. A−) diet as in Figure 1—figure supplement 1. Ileal Saa expression was quantified by
Q-PCR (primer sequences are given in Table 1). N = 3–5 mice per condition. (B) Ileal sections were stained with
anti-SAA antibody (‘Materials and methods’) and anti-rabbit IgG-Cy3 (red), and counterstained with DAPI (blue).
Scale bar = 50 µm. (C) Q-PCR determination of Saa expression levels in livers of mice on a normal or vitamin
A-deficient diet. N = 5 mice/condition. (D) Liver sections were stained with anti-SAA antibody and anti-rabbit
IgG-Cy3 (red), and counterstained with DAPI (blue). Scale bars = 50 µm. (E and F) Analysis of SAA expression in
HepG2 cells. Cells were cultured in retinoid-free medium and then treated with IL-1β and IL-6 and/or 1 μM retinol
(E) or 100 nM retinoic acid (F). SAA expression was determined by Q-PCR. N = 3 independent experiments.
Mean ± SEM is plotted. nd, not detected. *p < 0.05; **p < 0.01; ***p < 0.001. p values were determined by
two-tailed Student's t test.
DOI: 10.7554/eLife.03206.004
The following figure supplements are available for figure 1:
Figure supplement 1. Intestinal Saa1 and Saa2 are differentially regulated by dietary vitamin A.
DOI: 10.7554/eLife.03206.005
Figure supplement 2. Retinoid supplementation stimulates Saa expression in intestine and liver.
DOI: 10.7554/eLife.03206.006

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 Research article Biophysics and structural biology | Immunology

Figure 2. Human and mouse SAAs bind retinol. (A) Retinol exhibits intrinsic fluorescence that is enhanced upon
binding to proteins through energy transfer from tryptophan residues. All-trans-retinol was titrated into mSAA3 and
fluorescence emission was monitored following excitation at 348 nm. The chemical structure of retinol is shown.
(B) All-trans-retinol was titrated into hSAA1, mSAA1, mSAA3, human transferrin (hTfr; negative control), and apolipo-
protein A1 (ApoA1; negative control). Binding was quantified by monitoring retinol fluorescence at 460 nm following
excitation at 348 nm as in (A). Plots are representative of five independent experiments. (C) Retinoic acid lacks
intrinsic fluorescence, but can quench intrinsic protein fluorescence due to energy transfer from tryptophan residues
(Cogan et al., 1976). All-trans-retinoic acid was titrated into mSAA3 and fluorescence quenching was monitored
following excitation at 296 nm. The chemical structure of retinoic acid is shown. (D) All-trans-retinoic acid was
titrated into hSAA1, mSAA1, mSAA3, hTfr, and ApoA1. Fluorescence emission was monitored at 334 nm with
excitation at 296 nm as in (C). Plots are representative of three independent experiments. (E) Kds were calculated
from the binding assay data plotted in (B) and (D) and were derived from three independent experiments. nd, not
determined. Additional ligand binding measurements are provided in Figure 2—figure supplements 1 and 2.
DOI: 10.7554/eLife.03206.007
The following figure supplements are available for figure 2:
Figure supplement 1. Retinol and retinoic acid binding to human retinol binding protein 4 (hRBP4).
DOI: 10.7554/eLife.03206.008
Figure supplement 2. Additional ligand binding studies on human and mouse SAAs.
DOI: 10.7554/eLife.03206.009

(Figure 2C). Titration of all-trans retinoic acid yielded Kds of 268 and 224 nM for retinoic acid binding
to hSAA1 and mSAA3, respectively (Figure 2D,E), which are similar to binding affinities calculated for
human RBP binding to retinoic acid (Cogan et al., 1976) (Figure 2—figure supplement 1). There was

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weak binding of retinoic acid to mSAA1 and we were unable to calculate a Kd for the interaction
(Figure 2D,E). Thus, while hSAA1 and mSAA3 bind both retinol and retinoic acid, mSAA1 selectively
binds retinol. mSAA1 also showed weak binding to other retinoids, including β-carotene and retinyl
acetate, while hSAA1 bound these compounds with Kds of 497 and 347 nM, respectively, and mSAA3
bound β-carotene with a Kd of 159 nM (Figure 2—figure supplement 2A,B). All SAA isoforms bound
weakly to retinyl palmitate (Figure 2—figure supplement 2C). Since long chain retinyl esters (such as
retinyl palmitate) are the major form of stored retinoid in the liver (Vogel et al., 1999), this suggests
that SAAs do not transport retinoids for storage. Although a role for SAAs in cholesterol transport and
metabolism has been proposed (van der Westhuyzen et al., 2005), we found that cholesterol was
unable to competitively inhibit retinol binding to SAAs (Figure 2—figure supplement 2D).
To test whether SAAs also associate with retinol in vivo, we sought to purify SAAs from mouse tis-
sues and assay for the presence of associated retinol. SAAs were difficult to purify from the mouse
intestine due to the presence of large amounts of contaminating protein, even under conditions where
expression of SAAs was maximally induced. However, SAAs constitute a high proportion of serum
protein during acute systemic infection (McAdam and Sipe, 1976; Zhang et al., 2005). We were
therefore able to use size exclusion chromatography to recover a SAA-enriched fraction from the
sera of mice infected intraperitoneally with Salmonella typhimurium for 24 hr (Figure 3—figure
supplement 1A–C). Mass spectrometry revealed that the SAA-enriched protein fraction was devoid
of other known retinol binding proteins (Figure 3—figure supplement 1D). Liquid chromatography
tandem mass spectrometry (LC-MS/MS) indicated the presence of retinol in the SAA-enriched
fraction (Figure 3, Figure 3—figure supplement 2A–C) in a molar ratio of ∼1 mol retinol/4 mol
SAA (Figure 3, inset). In these analyses, retinoic acid was not detected, and retinol was not detected
in the equivalent serum fraction recovered from Saa1/2−/− mice (Eckhardt et al., 2010) (Figure 3),
suggesting that the retinol was preferentially associated with SAA.

Mouse SAA3 forms a tetramer with a hydrophobic central channel

SAAs lack sequence homology to the two known families of retinol binding proteins: cellular retinol
binding proteins (CRBP) and serum retinol binding proteins (RBP) (Blaner, 1989; Noy, 2000). Thus, the
three-dimensional structures of CRBP and RBP proteins (Newcomer et al., 1984; Cowan et al., 1993)
provide no direct insight into the structural basis for retinol binding by SAAs. To understand how SAAs
bind retinol, we therefore determined the three-dimensional structure of recombinant mSAA3 by
X-ray crystallography. The protein was crystallized in a P62 space group with two subunits in the asym-
metric unit, and the structure was determined to a resolution of 2 Å by single-wavelength anomalous
dispersion (SAD) phasing using a selenomethionyl-derivatived crystal (Figure 4A; Table 2). The crystal
structure reveals that mSAA3 is highly α-helical (Figure 4A), as predicted on the basis of its primary
sequence (Figure 4—figure supplement 1) (Stevens, 2004). The structure is very similar to the
recently determined structure of human SAA1.1 (Lu et al., 2014), an isoform that has a marked
tendency to form pathogenic amyloid fibrils (Yu et al., 2000). Like the SAA1.1 structure, the mSAA3
structure consists of four α-helices, designated α1-4 from the N- to the C-termini, forming a cone-
shaped four-helix bundle with a comparatively longer α1. The helices form two sets of antiparallel
helices, α1-α2 and α3-α4, connected by a very short loop (Figure 4A). The monomer is stabilized by
an extensive network of hydrogen bonding interactions among conserved residues and water mole-
cules in the interior of the monomer. As in the SAA1.1 structure, the C-terminal tail wraps around the
helix bundle making a number of hydrogen bonding interactions that add to monomer stability, under-
scoring the importance of the C-terminal tail.
Size exclusion chromatography and cross-linking experiments showed that mSAA3 forms a tetramer
in solution (Figure 5A,B). Consistent with these findings, analysis of the mSAA3 crystal structure using
the Protein Interfaces, Surfaces and Assemblies (PDBePISA) server (http://www.ebi.ac.uk/msd-srv/
prot_int/) yielded a tetrameric quaternary structure (Figure 4B; Table 3). This is in contrast to the hexa-
meric structure derived for SAA1.1 (Lu et al., 2014). There are several potential reasons for the discrep-
ancy in the oligomeric structures. First, it has been suggested that different SAAs can adopt different
oligomeric states (Wang et al., 2002, 2011). SAA1.1 was also observed to produce a ∼43 kDa species
in solution (Lu et al., 2014), suggesting that SAA1.1 may in part adopt a tetrameric state. Second,
SAA1.1 has a more hydrophobic N-terminus than mSAA3, which is thought to be a determinant of
amyloidogenicity (Yu et al., 2000; Lu et al., 2014). Third, the crystallized SAA1.1 protein retained the
hexa-histidine tag (Lu et al., 2014), which may have contributed to the difference in oligomeric state.

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Figure 3. Serum SAA is associated with retinol in vivo. Wild-type or Saa1/2−/− mice were infected intraperitoneally
with S. typhimurium and serum was collected 24 hr later. The serum was fractionated by size exclusion chromatog-
raphy and a major SAA-containing fraction from wild-type mice was identified by Western blot (Figure 3—figure
supplement 1). The SAA-containing fraction and the equivalent serum fraction from Saa1/2−/− mice were
hexane-extracted and analyzed by LC-MS/MS against retinol and retinoic acid standards. Additional support for
the identification of retinol is provided in Figure 3—figure supplement 2. The LC-MS/MS chromatograms of
daughter ion 93 are shown. mol SAA/mol retinol is shown in the inset. Data are representative of duplicate
experiments with triplicate samples in each experiment.
DOI: 10.7554/eLife.03206.010
The following figure supplements are available for figure 3:
Figure supplement 1. Size-exclusion chromatography and mass spectrometry analysis of SAA-containing serum
fractions.
DOI: 10.7554/eLife.03206.011
Figure supplement 2. Mouse SAA is associated with retinol in the serum following infection.
DOI: 10.7554/eLife.03206.012

The mSAA3 tetramer is formed by two sets of tightly associated dimers (Figure 4B,C). The dimers
pack against each other through an aromatic interface formed by W71, as well as a non-polar inter-
action involving V75 residues (Figure 4C,E). The dimer is formed by two identical chains oriented
pseudo-anti-parallel to each other, and is held together by two pairs of tight hydrogen bond interac-
tions between K74 and D78 residues on oppositely oriented α3 helices (Figure 4C,D). The interac-
tion is also supported by a set of aromatic interactions between F99 and W103 of the α4 helices
(Figure 4C,D). These interactions result in tightly held α3 helices composed primarily of non-polar resi-
dues, thus forming the inside hollow pocket of the tetramer. The hollow pocket is surrounded by the
remaining helices, creating a hydrophobic interior that is protected from the external aqueous
environment.

The mouse SAA3 central channel is predicted to accommodate retinol

Retinol is a highly apolar lipid-like molecule consisting of a β-ionone ring, an isoprenoid tail, and a
hydroxyl group. Thus, it requires a non-polar environment for transport among tissues and within cells.

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Figure 4. Structure of mSAA3 and molecular contacts within the tetrameric unit. (A) Structure of the mSAA3
monomer (side view), with helices and termini labeled. (B) Top view of the tetrameric mSAA3 structure. Chains
forming dimer pairs are colored cyan and magenta. In (C), helices α1-4 and the N- and C-termini of two monomers
are labeled. Residues that make dimer contacts are shown as green sticks while residues involved in tetramer
stabilization are shown as yellow sticks. (D and E) Magnified regions of a dimer interface (D) and tetramer interface
(E) are shown. Views are slightly rotated so that the interactions can be clearly visualized. Crystal structure data
collection and refinement statistics are provided in Table 2; alignments of mouse and human SAAs are shown in
Figure 4—figure supplement 1; parameters from the protein Interfaces, Surfaces, and Assemblies (PISA) analysis
showing a tetrameric state are provided in Table 3.
DOI: 10.7554/eLife.03206.013
The following figure supplement is available for figure 4:
Figure supplement 1. Sequence alignment of mouse and human SAAs.
DOI: 10.7554/eLife.03206.014

Structures of known retinol binding proteins, including serum RBP, indicate that these proteins consist
mainly of β-sheet secondary structures forming a β-barrel tertiary structure, with the retinol molecule
held in an interior non-polar binding pocket (Newcomer et al., 1984). In contrast, mSAA3 is α-helical
and oligomerizes to form a hollow, largely non-polar interior that could serve as a binding pocket for
a non-polar small molecule (Figure 6A). We were unable to obtain mSAA3 crystals with the bound

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Table 2. Crystal structure data collection and retinol ligand as retinol is highly unstable (Barua
refinement statistics and Furr, 1998) and the crystals required several
weeks to grow. However, a ligand docking anal-
Data collection
ysis using SwissDock (Grosdidier et al., 2011)
  Space group P62 indicated that retinol can be docked in this
  Cell dimensions (Å) a = b = 78.33, c = 62.32 hydrophobic pocket with favorable free energy
α = β = 90o, γ = 120o (∼ −7 kcal/mol) and FullFitness values (Zoete
  Wavelength (Å) 0.9794 et al., 2010) (∼ −2400 kcal/mol) (Figure 6B–D).
Consistent with this prediction, introducing a
 Rsym or Rmerge (%) 8.4
Trp71Ala (W71A) mutation in the mSAA3 hydro-
  Resolution (Å)* 50–2.05 (2.09–2.05)
phobic core reduced the affinity of mSAA3 for
 I/σI 19.19 (3.23) retinol (Figure 6E). Thus, the mSAA3 structure
  Completeness (%) 99.8 (97.3) supports our biochemical data showing a ret-
 Redundancy 6.2 (5.4) inol binding function for SAAs and explains how
Refinement mSAA3 could bind retinol.
  No. reflections 12,206
Discussion
  Resolution (Å)* 39.17–2.06 (2.14–2.06)
Numerous biochemical, physiologic, and epide-
 Rwork/Rfree 0.17/0.21 (0.16/0.19) miologic studies have shown that vitamin A and
  No. atoms its derivative retinol are essential for the develop-
  Protein 1608 ment of robust immunity (Stephensen, 2001).
  Ligand/ion 3 Retinol and retinoic acid are critical for the devel-
opment of innate and adaptive immunity (Lawson
  Water 61
and Berliner, 1999; Mucida et al., 2007; Hall
  R.m.s. deviations
et al., 2011; Spencer et al., 2014), and also pro-
   Bond lengths (Å) 0.0077 mote maintenance and repair of epithelial bar-
   Bond angles (°) 0.932 riers (Osanai et al., 2007). However, a prominent
response to acute infection is the marked decline
*Highest resolution shell is shown in parenthesis.
in serum RBP (Rosales et al., 1996), which para-
DOI: 10.7554/eLife.03206.015
doxically occurs at a time of increased demand
for retinol to support development of immunity
and barrier defense. Thus, it has been unclear how retinol is transported among tissues following an
acute microbial challenge.
We propose that SAAs fulfill this role, supported by several lines of evidence. First, SAAs are
strongly induced by microbial exposure at sites of retinol uptake (intestine) and storage (liver), and are
present at high levels in the circulation following microbial challenge (Chiba et al., 2009; Ivanov et al.,
2009; Reigstad et al., 2009). Second, we have shown that SAAs bind retinol at nanomolar affinity in
vitro, and that serum SAAs circulate in association with retinol. Third, the three dimensional structure
of mSAA3 exhibits a hollow hydrophobic binding pocket, providing structural insight into how SAAs
bind retinol.
Although the precise tissue targets of circulating retinol-bound SAAs remain under investigation,
several observations support the idea that SAAs promote immunity to infection. First, Saa1/2−/− mice
exhibit increased susceptibility to chemically-induced colitis in mice (Eckhardt et al., 2010), suggest-
ing that SAAs contribute to intestinal immunity. Second, studies in zebrafish show that commensal
microbiota stimulate neutrophil migration through induction of SAA (Kanther et al., 2013). Third, we
found that intraperitoneal infection of Saa1/2−/− mice with S. typhimurium resulted in higher bacterial
loads in liver and spleen as compared to wild-type mice (Figure 7A,B), suggesting that SAAs also
contribute to systemic immunity.
SAA4 is an SAA isoform that is expressed in the livers of healthy, non-infected mice and humans
(de Beer et al., 1991, 1994, 1995). SAA4 circulates at concentrations that are markedly lower than
those observed for SAA1 and 2 following acute infection (de Beer et al., 1995) but are similar to the
concentrations of RBP in uninfected individuals (Willett et al., 1985; Friedman et al., 1986). SAA4 is
54–56% homologous to SAA1, 2, and 3, and retains the hydrophobic amino acids that are predicted
to line the hydrophobic binding pocket in SAA3. Further, homology modeling using the mouse SAA3
structure and the mouse SAA4 sequences yields a SAA4 model with a highly similar predicted struc-
ture (alignment score of 0.1 and a Global Model Quality Estimate of 0.73). Thus, the possibility that

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 Research article Biophysics and structural biology | Immunology

Figure 5. Mouse SAA3 is tetrameric in solution. (A) Size exclusion chromatography profile of purified mouse SAA3
on a Superdex 75 10/300 GL column. Elution of standards (BioRad) is shown in the top panel and elution of SAA3
is shown in the bottom panel. mSAA3 elutes at a position consistent with a tetramer (monomer is 12.2 kDa).
(B) Cross-linking analysis of mSAA3. Purified mSAA3 was cross-linked with glutaraldehyde and analyzed by SDS-PAGE.
DOI: 10.7554/eLife.03206.016

SAA4 is a retinol binding protein that functions to transport retinol in healthy, non-infected animals will
be a subject for future investigation.
SAA1, 2, and 3 are markedly induced in the intestinal epithelium by the microbiota (Ivanov et al.,
2009; Eckhardt et al., 2010; Reigstad and Bäckhed, 2010), and thus our findings may provide insight
into how the intestinal microbiota regulates host immunity and inflammation. Previous studies have
suggested that SAAs promote Th17 cell development in response to specific components of the
microbiota, such as segmented filamentous bacteria (Ivanov et al., 2009). Consistent with a function
for SAAs in retinol binding and transport, retinol/retinoic acid is required to elicit Th17 cell responses
to infection and mucosal vaccination (Hall et al., 2011). A key question is whether there are tissue-
specific effects of intestinal epithelial SAAs, or whether the intestinal SAAs enter the circulation with
bound retinol acquired directly from the diet. For example, intestinal epithelial SAAs could be involved
in the direct delivery of retinol from epithelial cells to underlying immune cells in the lamina propria, or
from epithelial cells to mucosal lymphoid tissues.
Altogether, our results provide insight into
the biological function of SAAs, reveal a new pro-
Table 3. Parameters from the Protein Interfaces, tein architecture that supports retinol binding,
Surfaces, and Assemblies (PISA) analysis and suggest how retinol is transported among
Parameter Value cells and tissues during infection. These findings
Multimeric state 4 may prove useful in designing new strategies for
enhancing resistance to infection and/or control-
Composition A2B2
ling inflammation during disease.
Dissociation pattern 2(AB)
Surface area, Å2 19485.8
Materials and methods
Buried area, Å 2
6125.7
ΔGintrinsic, kcal/mol −79.8 Animals
ΔGdiss, kcal/mol 11.1 C57BL/6 wild-type mice were maintained in the
barrier at the University of Texas Southwestern
TΔSdiss, kcal/mol 12.6
Medical Center. Saa1/2−/− mice were obtained from
DOI: 10.7554/eLife.03206.017 Dr Frederick C de Beer (Eckhardt et al., 2010) at

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 Research article Biophysics and structural biology | Immunology

A B

90o
C D

E
100 Kd = 145 ± 73 nM
(relative units)
fluorescence

Kd = 496 ± 127 nM
50
mSAA3-wild-type
mSAA3-W71A
0
0 1 2 3 4 5
retinol (µM)

Figure 6. The mSAA3 tetramer forms a hollow hydrophobic binding pocket that can accommodate retinol. (A) A
surface rendering of the tetramer showing the interior cavity, with the electrostatic potential displayed using a color
gradient ranging from negative (red) to neutral (white) to positive (blue). The orientation is similar to that in Figure 4B.
(B–D) Different views of a retinol molecule docked in the putative ligand-binding pocket. (B) A semi-transparent
surface representation of the protein in the same orientation as Figure 4B, with a cartoon trace. Retinol atoms are
represented as green spheres. The views in (C) and (D) are rotated by approximately 90° in the horizontal plane
relative to (A) and (B), and (B) is rotated by approximately 90° in the vertical plane relative to (C). In (D), a surface
model of the protein is shown, sliced close to the binding pocket. Retinol atoms are shown as sticks. (E) Wild-type
or Trp71Ala (W71A) mutant mSAA3 was assayed for retinol binding as described in Figure 2. Representative plots
and Kds were calculated from the binding assay data and were derived from five independent experiments.
DOI: 10.7554/eLife.03206.018

the University of Kentucky and were maintained in the barrier at the University of Texas Southwestern
Medical Center. 6–12 weeks old mice were used for all experiments. Experiments were performed
using protocols approved by the Institutional Animal Care and Use Committees of the UT Southwestern
Medical Center.

Antibodies and reagents

Recombinant mSAA1 and 3 were expressed and purified as described below. hSAA1 protein was
from PeproTech (Rocky Hill, NJ) and resuspended as recommended. The protein is a consensus

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 Research article Biophysics and structural biology | Immunology

Figure 7. Saa1/2−/− mice have higher bacterial burdens following S. typhimurium infection. 10 week old wild-type
and Saa1/2−/− mice were inoculated intraperitoneally with 10,000 cfu of S. typhimurium. Livers (A) and spleens
(B) were collected after 24 hr and analyzed for bacterial counts by dilution plating. Combined results from two
independent experiments are shown. Each point represents one mouse and geometric means are indicated.
Dotted line indicates limit of detection. **p < 0.01 using the Mann–Whitney test.
DOI: 10.7554/eLife.03206.019

SAA molecule corresponding to human apo-SAA1α except for the presence of an N-terminal methi-
onine and substitution of asparagine for aspartic acid at position 60 and arginine for histidine at posi-
tion 71. Anti-SAA antiserum was raised against purified recombinant mSAA1. Retinol, retinoic acid,
β-carotene, retinyl acetate, retinyl palmitate, and cholesterol were from Sigma-Aldrich (St. Louis, MO)
and were reconstituted into ethanol, DMSO, or dioxane, depending on the experiment. IL-1β and IL-6
were from Invitrogen.

Vitamin A depletion
Vitamin A-deficient (TD.09838) and control (∼20,000 IU vitamin A/kg; TD.09839) diets were purchased
from Harlan Laboratories (South Easton, MA). At day 10 of gestation, pregnant females were placed on
the standard diet or the vitamin A-deficient diet (Hall et al., 2011). Mothers and pups were maintained
on the diets until weaning, and pups stayed on the diet for two additional months prior to sacrifice.

In vivo retinoic acid reconstitution

The protocol was adapted from a previously described procedure (Hall et al., 2011). A total of 250 μg
of all-trans-retinoic acid (Sigma-Aldrich) was resuspended in 30 μl of biotechnology performance cer-
tified DMSO (Sigma-Aldrich). The suspension was administered daily by intraperitoneal injection to
vitamin A-deficient mice over the course of 3 days. 24 hr following the third injection, mice were sac-
rificed and tissues were harvested. Control mice received an injection of the DMSO vehicle.

Intestinal explant culture

Terminal ileum (5 cm) was collected from mice post-sacrifice and flushed with a solution of phosphate-
buffered saline with penicillin (100 units/ml) and streptomycin (100 μg/ml). Ileal segments were cul-
tured on equilibrated cell culture plate inserts (PIHA03050; Millipore) at 37°C and 95% oxygen for 6 hr
in Dulbecco's modified Eagle's medium (4 g/l glucose and L-glutamine; Invitrogen, Carlsbad, CA)
supplemented with 10% charcoal-stripped heat-inactivated fetal bovine serum (Gibco, Carlsbad, CA),
10% NCTC135 media (Sigma), 25 mM HEPES, 100 units/ml penicillin, 100 μg/ml streptomycin, and
either 0.1% DMSO or 1 μM retinol in 0.1% DMSO. After culture for 6 hr, segments were flash-frozen
and processed for total RNA extraction.

Microarray experiments
Total RNAs were isolated from mouse ileum using the Qiagen Midi-Prep RNA isolation kit. For each
condition, RNA was isolated from two independent groups of five to eight mice. The RNAs in each

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 Research article Biophysics and structural biology | Immunology

group were pooled and used to generate biotinylated probes for microarray analysis. Probes were
hybridized to Affymetrix Mouse Genome 430 2.0 GeneChips in the University of Texas Southwestern
Microarray Core.
To identify genes that are differentially expressed between germ-free and conventional mice,
we performed two-way comparisons between germ-free and conventional groups, with germ-free
samples designated as baseline. Raw data were imported into Affymetrix (Santa Clara, CA) GeneChip
software for analysis, and previously established criteria were used to identify differentially expressed
genes (Cash et al., 2006). Briefly, a twofold difference was considered significant if three criteria were
met: (1) the GeneChip software returned a difference call of increased or decreased; (2) the mRNA was
called present by GeneChip software in either germ-free or conventional samples; and (3) the differ-
ence was observed in duplicate microarray experiments. We performed a similar analysis to identify
genes that are differentially regulated between mice fed a normal diet vs those fed a vitamin A-deficient
diet. Finally, we identified 19 genes that were differentially regulated by colonization status and by
dietary vitamin A content. Signal intensity data for this group of 19 genes were converted to Z-scores
(z = (x − μ)/σ, where x = signal intensity, μ = mean signal intensity for all samples, and σ = SD across
all samples), which were visualized as heatmaps using Java TreeView software.

Quantitative PCR
Total RNA was isolated from homogenized tissues or cells using the Qiagen RNeasy RNA isolation kit.
Random primed cDNAs were assayed by SYBR Green-based real-time PCR using SAA-specific primers
as given in Table 1. Signals were normalized to 18S rRNA or Gapdh.

Immunofluorescence analysis
Zinc-fixed, paraffin embedded tissue sections were stained with anti-SAA antiserum raised against
purified recombinant mSAA1 and detected using a goat anti-rabbit IgG Cy3 conjugate (Biomeda).
Tissues were counterstained with DAPI and images were captured on a Zeiss AxioImager M1
Microscope.

Cell culture
HepG2 cells were purchased from ATCC. Cells were maintained in 1X DMEM, 10% FBS (or charcoal
stripped FBS), 1X Penstrep, 1X glutamax, and 1X sodium pyruvate. Cells were maintained at 5% CO2.
Prior to addition of retinoids, the cells were grown overnight in DMEM containing 10% charcoal-
stripped FBS (to removes retinoids) and were treated with 1 μM retinol or 100 nM retinoic acid, 10 ng/ml
of IL-1β, and 10 ng/ml IL-6.

Expression and purification of recombinant SAAs

Genes encoding mouse SAA1 and SAA3 (minus the signal sequence) were cloned into the pET28(a)+
expression vector between NdeI and BamHI restriction endonuclease sites, with an N-terminal hexa-
histidine tag followed by a thrombin cleavage site and a C-terminal stop codon. Proteins were expressed
in Escherichia coli BL21-CodonPlus (DE3)-RILP cells (Stratagene, La Jolla, CA) by induction with 0.4 mM
isopropyl-β-D-galactoside (IPTG) for ∼3 hr at 25°C for mSAA1, and at 37°C for mSAA3. Cells were har-
vested by centrifugation at 4500×g for 25 min at 4°C and re-suspended in lysis buffer (50 mM NaH2PO4,
500 mM NaCl, 10 mM imidizole for mSAA1 and 500 mM NaCl, 50 mM Tris pH 8.0, 10 mM imidazole, 15
mM β-mercaptoethanol for mSAA3). After sonication, decyl maltopyranoside (DM) (Avanti Polar Lipids,
Alabaster, AL) was added to a final concentration of 40 mM and incubated for ∼3 hr at 4°C. The mixture
was pelleted by centrifugation at 10,000×g for 30 min and the supernatant loaded onto a Ni2+ metal
affinity column (Qiagen, Valencia, CA) pre-equilibriated with 4 mM DM in lysis buffer. Non-specific con-
taminants were washed away with 25 mM imidazole in DM buffer and the protein was eluted in DM
buffer containing 300 mM imidazole. All subsequent buffers do not include detergent in order to com-
pletely remove detergent. The eluate was desalted with a HiTrap desalting column (GE Life Sciences,
Pittsburgh, PA) into a 200 mM NaCl buffer. Thrombin (Roche, Basel, Switzerland) was then added (∼1
unit/1.2 mg protein) and incubated overnight at 4°C. Undigested protein was removed by passing the
overnight digest over Ni2+ affinity matrix, collecting only the flow through. The eluate was concentrated
in a 3 K cutoff Amicon Ultra centrifugal device (Millipore, Billerica, MA) and further purified by size exclu-
sion chromatography on either a HiLoad Superdex 200 or a Superdex 75 (10/30) column (GE Life
Sciences, Pittsburgh, PA), in 100 mM NaCl, 20 mM Tris pH 8, 15 mM a β-mercaptoethanol and 5% glycerol.
For crystallization, mSAA3 was concentrated to ∼3 mg/ml.

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 Research article Biophysics and structural biology | Immunology

Mutagenesis
The mSAA3-W71A mutant was generated using the QuickChange II site-directed mutagenesis kit
(Agilent Technologies, Santa Clara, CA). Protein expression, purification, and retinol binding assays
were done as for wild-type mSAA3.

Binding assays
Steady state fluorescence was measured using a QuantaMaster 40 spectrofluorometer (Photon
Technology International, Edison, NJ) and FelixGX software program. For retinol titrations, samples
were excited at 348 nm and emissions monitored at 460 nm. For retinoic acid and other retinoids,
samples were excited at 296 nm and tryptophan quenching was monitored by emission at 334 nm.
Experiments on mSAA1 and mSAA3 were done in 25 mM Tris pH 8.0, 100 mM NaCl, and 4 mM decyl
maltopyranoside (DM). Experiments with hSAA1, human ApoA1, and human transferrin were conducted
in PBS. For cholesterol competition assays, saturating concentrations of retinol were added to hSAA1,
mSAA1, and mSAA3, and fluorescence quenching was monitored by emission at 334 nm. 10 μM
cholesterol was added to the assay and inhibition of fluorescence quenching by retinol was monitored.
All assays were done using a protein concentration of 0.5 μM.

S. typhimurium infections
Salmonella enterica Serovar Typhimurium (SL1344) was grown overnight in Luria Broth at 37°C. Mice
were infected intraperitoneally with 1 × 104 organisms per mouse. Mice were sacrificed after 24 hr and
tissues and serum were collected for experiments.

Size exclusion chromatography of serum SAA

Serum was pooled from 3–5 mice and 500 μl was separated by size-exclusion chromatography on a
Superdex 75 HiLoad 16/60 column (GE Life Sciences). Peak fractions were analyzed by SDS-PAGE and
stained with Coomassie Blue. Duplicate samples were analyzed by Western blot with anti-SAA anti-
body to identify peak fractions containing SAA protein.

Mass spectrometry analysis of retinol and retinoic acid

Retinoid extraction was modified and scaled from a previously described procedure (McClean et al.,
1982). SAA-containing fractions purified by size exclusion chromatography were pooled, added to an
equal volume of 1:1 1-butanol:acetonitrile, and vortexed for 60 s. 20 μl of 20.6 M K2HPO4 was added
for each 1 ml of pooled fractions. Samples were then vortexed 30 s and 5 ml of hexane per 1 ml sample
was added. Samples were vortexed for another 30 s and centrifuged at 1,000×g for 5 min and the top
organic phase was dried in a nitrogen evaporator (Organomation Associates, Berlin, MA). Samples
were prepared the day before the assay and stored at 80°C. Standard solutions were resuspended in
ethanol and prepared fresh for every use. Standard curves were generated by spiking retinol or retinoic
acid into 1 ml of 20 mM Tris pH 8.0, 100 mM NaCl and processed as for serum samples. Samples were
resuspended in 200 μl of acetonitrile before injection. Compound levels were monitored by LC-MS/MS
on an AB/Sciex (Framingham, MA) 4000 Qtrap mass spectrometer coupled to a Shimadzu (Columbia, MD)
Prominence LC after a 20 μl injection. The compounds were detected using electrospray ionization
(ESI) with the mass spectrometer in MRM (multiple reaction monitoring) mode by following the pre-
cursor to fragment ion transition 269.2 → 93.1 and 269.2 → 119 for retinol (pos. mode; [M-H2O]+) and
301.2 → 123.1 for retinoic acid (pos. mode; M+H+). An Agilent (Santa Clara, CA) Eclipse XDB C18
column (150 × 4.6 mm, 5 micron packing) was used for chromatography with the following condi-
tions: mobile phase A: acetonitrile:methanol:H2O:formic acid (55:33:12:.01); mobile phase B:
acetonitrile:formic acid (100:0.01). Over a total run time of 18 min, the following gradient was applied:
0 to 3 min 50% B; 3 to 10 min gradient to 100% B; 10 to 17 min 100% B; 17 to 18 min gradient to 50%
B. Stoichiometries of the SAA-retinol association were determined by quantifying serum retinol and
serum SAA. Total serum retinol was calculated based on peak areas from the mass spectrometer anal-
ysis in samples compared to a retinol standard curve. Serum SAA was quantified by Western blot
analysis with anti-SAA antiserum and densitometry.

Determination of the mSAA3 crystal structure

Crystals were grown by sitting-drop vapor diffusion at 20°C by mixing equal volumes of protein and
reservoir. An initial hit was obtained in 30–40% 2-methyl-2,4-pentanediol (MPD), 0.1 M sodium acetate
pH 4.5 after more than a month. Further refinement yielded better crystals at 75–80% MPD. Crystals

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 Research article Biophysics and structural biology | Immunology

were directly flash frozen, the MPD serving as a cryoprotectant. Crystals were of space group P62
with cell dimensions a = 78.327 Å, c = 62.319 Å and two subunit copies in the asymmetric unit.
Selenomethionyl-derivatived crystals were grown the same way as the native protein crystals, except
the culture media used was defined media with selenomethionine additive (Molecular Dimensions,
Altamonte Springs, FL) and purification buffers after the last Ni2+ column elution contained 10 mM DTT
(instead of β-mercaptoethanol) and 0.5 mM EDTA.
Data were collected at 100 K under a nitrogen gas stream at the Advanced Photon Source (APS)
beamlines 19ID or 23IDD of the Argonne National Laboratory. Single-wavelength anomalous disper-
sion (SAD) data were collected at the selenium K edge (0.9793 Å). Diffraction data were processed
with the HKL2000/3000 package (Otwinowski and Minor, 2013). Heavy atom substructure as well as
initial phases were obtained using the SAD pipeline in the PHENIX crystallographic software package
(Afonine et al., 2012). This was followed by manual model building in Coot (Emsley et al., 2010)
interspersed with iterative rounds of rigid body, simulated annealing and individual isotropic B-factor
refinement and finally TLS refinement in PHENIX. The structure was determined to a resolution of 2 Å.
Data collection and refinement statistics are summarized in Table 2.

mSAA3 cross-linking analysis

Glutaraldehyde (Sigma) was added to varying final concentrations (0.05%, 0.005%, and 0.0005% wt/vol)
to purified mSAA3 (0.5 mg/ml in PBS). The reaction mixtures were incubated for ∼30 min on ice,
quenched with 0.1 M Tris pH 8, and analyzed by SDS-PAGE.

Statistics
Statistical differences were calculated by the unpaired two-tailed Student's t test or Mann–Whitney
test using GraphPad Prism software. Results are expressed as the mean ± standard error of the mean
(SEM).

Acknowledgements
We thank Cassie Behrendt Boyd, Charmaine Clements, and Tess Leal for assistance with mouse experi-
ments, the staff at 19-ID and 23-IDD of the Advanced Photon Source (APS) for beamline access, Jun
Liao, Youxing Jiang and Nam Nguyen for beamline access and discussions, the UT Southwestern
Structural Biology Laboratory core for help with data collection and Diana Tomchick for advice on
crystallography. This work was supported by NIH R01 DK070855 (LVH), the Welch Foundation (I-1762
to LVH), a Burroughs Wellcome Foundation Investigators in the Pathogenesis of Infectious Diseases
Award (LVH), and the Howard Hughes Medical Institute (LVH). MGD was supported by a UNCF/Merck
Postdoctoral Fellowship and a Burroughs Wellcome Fund Postdoctoral Enrichment Program Award,
CMZ was supported by NIH Grant T32 AI005284, and SV was supported by Crohn's and Colitis
Foundation of America Fellowship Award. Results shown in this report are derived from work performed
at Argonne National Laboratory, Structural Biology Center at the APS. GM/CA at the APS has been
funded in whole or in part with Federal funds from the National Cancer Institute (Y1-CO-1020) and the
National Institute of General Medical Sciences (Y1-GM-1104). Argonne is operated by UChicago
Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental Research
under contract DE-AC02-06CH11357. Coordinates of the crystallographic structure of mSAA3 have
been deposited in the PDB with accession code 4Q5G.

Additional information
Funding
Funder Grant reference number Author
Welch Foundation  I-1762 Lora V Hooper
Howard Hughes Medical Lora V Hooper
Institute
National Institutes of R01 DK070855 Lora V Hooper
Health 
Burroughs Wellcome Fund Minority Enrichment Mehabaw G Derebe
Program

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 Research article Biophysics and structural biology | Immunology

Funder Grant reference number Author

UNCF/Merck Postdoctoral Mehabaw G Derebe
Fellowship
Crohn's and Colitis Foundation Career Development Award Shipra Vaishnava
of America 
National Institutes of Health  T32 AI005284 Clare M Zlatkov
Burroughs Wellcome Fund Investigators in the Lora V Hooper
Pathogenesis of
Infectious Diseases

The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication.

Author contributions
MGD, CMZ, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting
or revising the article; SG, Acquisition of data, Analysis and interpretation of data, Drafting or revising
the article; KAR, SV, GED, JBMM, NSW, Acquisition of data, Analysis and interpretation of data; LVH,
Conception and design, Analysis and interpretation of data, Drafting or revising the article

Ethics
Animal experimentation: Animal subjects research approved by all animal experiments were approved
by the Institutional Animal Care and Research Advisory Committee at the University of Texas
Southwestern Medical Center, and the approved animal protocol number is 2011-0197. The institutional
guidelines for the care and use of laboratory animals were followed.

Additional files
Major dataset
The following dataset was generated:

Database, license,
and accessibility
Author(s) Year Dataset title Dataset ID and/or URL information
Derebe MG, Hooper LV 2014 Crystal Structure of mouse 4Q5G; http://www.rcsb.org/ Publicly available at the
Serum Amyloid A3 pdb/search/structidSearch. RCSB Protein Data Bank
do?structureId=4Q5G (http://www.rcsb.org/
pdb/home/home.do).

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